The chemokines constitute a diverse group of small secreted basic proteins, that regulate the chemotactic migration and activation of a number of different leukocytes, particularly in the context of activation of the immune response during inflammatory conditions.
Examples of cells that have been shown to chemotactically respond to and become activated by the chemokines are neutrophils, eosinophils, basophils, monocytes, macrophages, as well as B lymphocytes and different types of T lymphocytes (Oppenheim, J. J., et al. (1991) Annu.Rev.Immunol. 9, 617-48; Miller, M. D. & Krangel, S. K. (1992) Crit.Rev.Immunol. 12(1,2) 17-46; Baggiolini, M., et al. (1994) Adv.Immunol. 55, 97-179).
The chemokines can be classified into two major groups based on the pattern of cysteinyl residues participating in disulfide bond formation in mature proteins. The first group, the CXC chemokines, or the a-chemokines, are characterized by the occurrence of two cysteinyl residues in the amino-terminal region, between which a different amino acid residue is positioned.
The second group, the CC chemokines, or the b-chemokines, are characterized by the occurrence of two adjacent cysteinyl residues occurring in the amino-terminal region.
A third, minor group of chemokines, represented by lymphotactin that has been isolated from mice and humans (Kelner, G. S., et al.(1994) Science 266, 1395-9; Kennedy, J., et al. (1995) J.Immunol. 155,203-9), is characterized by the occurrence of only two cystein residues, presumably forming a single disulfide bond in the mature protein. So far, these are the only representatives of the so-called c-type chemokines.
The CXC chemokines act primarily on neutrophils, in particular those CXC chemokines that carry the amino acid sequence Glu-Leu-Arg on their amino terminus. Examples of CXC chemokines that are active on neutrophils, are interleukin-8 (IL-8), GRO-.alpha., -.beta., and -.gamma., NAP-2, ENA-78, and GCP-2.
The CC chemokines act on a larger variety of leukocytes such as monocytes, macrophages, eosinophils, basophils, as well as T and B lymphocytes. Examples of these are MCP-1, MCP-2, MCP-3, MIP-1.alpha., MIP-1.beta., eotaxin, RANTES, I-309. The latter carries two additional cysteinyl residues probably forming a third disulfide bond in the mature protein.
MCP-1, or monocyte chemoattractant protein 1, was originally purified from phytohemagglutinin stimulated human lymphocytes (Yoshimura, T. et al. (1989) J.Immunol. 142, 1956-62), a human glioma cell line (Yoshimura, T., et al (1989) J.Exp.Med. 169, 1449-59), and the human myelomonocytic cell line THP-1 (Matsushima, K., et al. (1989) J.Exp.Med. 169, 1485-90). MCP-1 has also been called MCAF, LDCF, GDCF, HC11, TSG-8, SCYA2, and A2. Molecular cloning of the cDNA encoding MCP-1 (Furutani, Y., et al. (1989) Biochem.Biophys.Res.Comm. 169. 249-55; Rollins, B. J., et al. (1989) Mol. Cell. Biol. 9, 4687-95; Chang, H. C., et al. (1989) Int. Immunol. 1, 388-97) revealed an open reading frame of 99 amino acids, including a signal peptide of 23 amino acids.
MCP-1 is produced by monocytes, and a variety of tissue cells, such as endothelial cells, epithelial cells, fibroblasts, keratinocytes, synovial cells, mesangial cells, osteoblasts, smooth muscle cells, as well as by a multitude of tumour cells (Baggiolini, M., et al. (1994) Adv.Immunol. 55, 97-179, and references therein).
Its expression is stimulated by several types of pro-inflammatory agents, such as IL-1.beta., TNF-.alpha., IFN-.gamma., lipopolysaccharide, and GM-CSF. MCP-1 is suggested to play an important role in the pathogenesis of atherosclerosis. Macrophages that are loaded with lipids, so-called foam cells, comprise the majority of cells in atherosclerotic lesions. It is suggested that active monocyte recruitment through MCP-1 from these cells and from activated endothelium plays a central role in the formation of fatty streaks and atherosclerotic plaques (Yla-Herttuala, S., et al. (1991) Proc.Natl.Acad.Sci.USA 88(12), 5252-6; Schwartz, C. J., et al. (1993) Am. J. Cardiol. 71(6), 9B-14B;Takeya, M. (1993) Hum. pathol. 24(5), 534-9).
In the synovial fluid and plasma of patients with rheumatoid arthritis, the concentration of MCP-1 has been shown to be increased, as compared to other arthritic diseases, and the main source of this is macrophages which constitutively express MCP-1. In the rheumatoid synovium, MCP-1 together with other inflammatory cytokines such as IL-1.beta., IL-6, IL-8 and TNF-.alpha., contribute to the immunopathogenesis of rheumatoid arthritis (Koch, A. E. (1992) J.Clin.Invest. 90, 772-79; Hosaka, S., et al. (1994) Clin.Exp.Immunol. 97, 451-7; Akahoshi, T., et al. (1993;) Arthritis.Rheum. 36, 762-71; Harigai, M., et al. (1993) Clin.Immunol.Immunopathol. 69, 83-91).
The mononuclear-phagocyte dependent lung injury mediated by IgA immune complexes is characterized by mononuclear and phagocytic cell accumulation, and has been shown to be largely dependent on the expression of MCP-1 (Jones, M. L., et al. (1992) J.Immunol. 149, 2147-54). Similarly, MCP-1 seems to play an important role in the pathogenesis of idiopathic pulmonary fibrosis and sarcoidosis (lyonaga, K., et al. (1994) Hum.Pathol. 25, 455-63; Car, B. D., et al (1994) Am.J.respir.Crit.Care.Med. 149, 655-9).
In animal models, MCP-1 has been shown to be expressed in the brain after focal ischemia (Kim, J. S., (1995) J.Neuroimmunol. 56, 127-34; Wang, X., et al. (1995) Stroke 26, 661-5), and during experimental autoimmune encephalomyelitis (Hulkower, K., et al. (1993) J.Immunol. 150, 2525-33; Ransohoff, R. M., et al. (1993) 7, 592-600). MCP-1 may be an important cytokine that targets mononuclear cells in the disease process illustrated by these animal models, such as stroke and multiple sclerosis.
In psoriatic lesions, MCP-1 seems to regulate the interaction between proliferating keratinocytes and dermal macrophages, and can be located above the dermal/epidermal junction. In addition to IL-8, which is important for the neutrophilic infiltration into these types of lesions, MCP-1 may serve to recruit mononuclear cells (Schroder, J. M. (1992) Arch. Dermatol. Res 284 Suppl 1, S22-6; Gillitzer, R., et al (1993) J.Invest.Dermatol. 101, 127-31).
MCP-1 seems to be involved in the control of mononuclear cell infiltration found in many solid tumors. A correlation between tumor associated MCP-1 production and the number and proliferative activity of tumor associated macrophages has been demonstrated (Walter, S., et al. (1991) Pathobiology 59(4), 239-42; Mantovani, A., et al. (1994) Adv.Exp.Med.Biol. 351, 47-54). Using transplanted sarcoma cells in mice, it has been demonstrated that cells expressing high levels of MCP-1 grow more slowly, and that this is related to the number of tumor associated macrophages (Walter, S., et al. (1991) Int.J.Cancer, 49, 431-5). Similarly, murine colon carcinoma cells engineered to express MCP-1, display reduced metastatic potential (Huang, S., et al. (1994) Cancer Immunol. Immunother. 39, 231-8).
MCP-1 is a powerful chemoattracting and activating factor for monocytes, inducing chemotaxis, calcium flux, and the respiratory burst, showing activity in the picomolar range (Yoshimura, T. & Leonard, E. J. (1990) J.Immunol. 154, 292-97; Zachariae, C. O. C., et al. (1990) J.Exp.Med. 171, 2177-82; Rollins, B., et al. (1991) Blood 78, 1112-6; Vaddi, K. & Newton, R. C. (1994) J.Leukoc.Biol. 55, 756-62). MCP-1 also upregulates CD11b/CD18 and CD11c/CD18 in a transient time course, which is likely to facilitate trans-endothelial migration during inflammation Oiang, Y, et al. (1992) J.Immunol. 148, 2423-8; Vaddi, K. & Newton, R. C. (1994) J. Immunol. 153, 4721-32).
It has recently become evident that in addition to monocytes, MCP-1 acts on CD4+ and CD8+ T lymphocytes as a chemoattractant both in vitro and in vivo (Loetscher, P., et al. (1994) FASEB J. 8, 1055-60; Carr, M. W., et al. (1994) Proc.Natl.Acad.Sci.USA 91, 3652-6; Taub, D. D., et al. (1995) 95, 1370-6). Natural killer cells that have been stimulated by interleukin-2, are also subject to chemotaxis by MCP-1 (Maghazachi, A A., et al. (1994) J.Immunol. 153, 4969-77; Allaven, P., et al. (1994) Eur.J.Immunol. 24, 3233-6. This underscores the importance of this chemokine in the recruitment of effect of cells into inflammatory lesions.
In addition to the effects on monocytes and T lymphocytes, MCP-1 is a moderate chemoattractant and potent activator of allergy mediator release, such as histamine and leukotrienes, from basophils (Kuna, P., et al. (1992) J.Exp.Med. 175, 489-93; Bischoff, S. C., et al. (1992) J.Exp.Med. 175, 1271-7; Bischoff, S. C., et al. (1993) Eur. J. Immunol. 23, 761-7). In contrast to basophils, another granulocyte implicated in allergic inflammatory lesions, the eosinophil, does not respond to MCP-1 (Rot, A., et al (1992) J.Exp.Med. 176, 1489-95).
There exists one receptor for MCP-1, that seems to be expressed in two slightly different forms due to alternative splicing of the mRNA encoding the carboxy-terminal region, MCP-1-RA and MCP-1RB (Charo, I. F., et al. (1994) Proc.Natl.Acad.Sci.USA, 91, 2752-56).
These receptors (CCR2) are expressed in monocytes, myeloid precursor cells and activated T lymphocytes (Myers,S. J.,et al, 1995. J. Biol. Chem., 270, 5786-5792, Qin, S. et al. 1996. Eur. J. Immunol. 26, 640-647). They provide an effective means of defining the molecular basis of chemokine-receptor interactions and of understanding MCP-1's role in the regulation of monocyte/macrophage infiltration in a variety of disease processes.
The MCP-1 receptor belongs to the seven transmembrane-type of proteins, and is homologous to the receptors for MIP-1.alpha./RANTES (CC-CKR1; Neote, K. et al. (1993) Cell 72, 415-25) and Interleukin-8/GRO (Holmes, W. E., et al. (1991) Science 253, 1278-80; Murphy, P. M. & Tiffany, H. L. (1991) Science 253, 1280-3). The dissociation constant of MCP-1 to the receptor transfected into HEK-293 cells is 0.26 nM which is in agreement with values measured on monocytes (Myers, S. J. et al. (1995) J.Biol.Chem. 270, 5786-92; Van Riper, G. et al. (1993) J.Exp.Med. 177, 851-6). Activation of the MCP-1RB receptor on transfected HEK-293 cells by MCP-1 inhibits adenylyl cyclase at a concentration of 90 pM, and mobilizes intracellular calcium at slightly higher concentrations, seemingly independent of phosphatidyl inositol hydrolysis. The effects on adenylyl cyclase and intracellular calcium release are strongly inhibited by pertussis toxin, implying the involvement of Gi type heterotrimeric G-proteins in the signal transduction (Meyers S. J. et al, (1995) J. Biol. Chem. 270, 5786-92).
The recent description of chemokine receptors as HIV-1 coreceptors and of chemokines as neutralizing agents of HIV-1 infection, assigns these molecules a key role in HIV-1 pathogenesis (Doranz, B. J. et al. 1996, Cell 85, 1149-1158; Feng, Y et al. 1996. Science 272, 872-877; Deng, H et al, 1996. Nature 381, 661-666; Cocchi, F et al. 1995, Science 270, 1811-1815; Choe, H et al. 1996, Cell 85, 1135-1148; Alkhatib, G et al, 1996. Science 272,1955-1958). Specific tools are essential to aid in unravelling the manner in which chemokines and HIV-1 interact with the chemokine receptors, to determine which receptors are important in directing different HIV-1 strains to different peripheral blood mononuclear cell (PBMC) populations, and at what stage (binding, desensitization, signal transduction) this interaction takes place.
WO 95/19436, a patent application filed in the name of The regents of the University of California, describes an isolated DNA sequence that codes on expression for MCP-1 receptor. It is also mentioned that an antagonist of the MCP-1 receptor could be identified by expression of the N-terminal domain of MCP-1 receptor and detection of a loss in binding of the MCP-1 receptor domain. A pharmaceutical composition is claimed which comprises the MCP-1 receptor antagonist as identified by the disclosed method. No such identification is performed and no antagonists are isolated.
We have generated mAb specific for the CCR2 chemokine receptor by immunizing mice with synthetic peptides corresponding to several extracellular receptor domains. We describe the generation of mAb capable of specifically recognizing native CCR2 receptor. Analysis of CCR2 expression on human PBMC and tonsil cells shows its expression in B cells, which is thus added to its known expression in monocytes and activated T cells. This would suggest a role for MCP-1 in B cells. These mAb were characterized by their ability to block and/or mimic MCP-1 activity, based on chemotaxis and Ca.sup.2+ induction in human monocytes and monocytic cell lines. Using these mAb, we define CCR2 regions critical for ligand binding and for eliciting a response through this receptor; we show a dissociation between these two activities that may help to unravel the complex mechanisms involved in chemokine signaling as well as the specificity relationship of these types of receptors. Based on the ability of these mAb to either trigger chemokine receptors or to block chemokine responses, we have outlined a model that takes into account our present knowledge of chemokine responses.
In contrast to IL-8 receptor neutralizing antibodies, which are directed to the NH.sub.2 terminal region (37), our mAb with antagonist activity (MCPR-04 and MCP R-05) map to the third extracellular loop region of the CCR2 receptor. Another mAb with similar peptide specificity (MCP R-03), although it binds to the receptor, do not block the MCP-1 activity. The neutralizing activity of these antibodies is thus limited to the recognition of a few key residues within this region, which play a critical role in chemokine binding or in the modulation of chemokine activity. Earlier studies conclude on the importance of the chemokine receptor NH.sub.2 terminal domain for the IL-8R, a member of the CXC chemokine receptor family. Several explanations may account for this difference. First, the structural features controlling CC chemokine interaction with its receptor may be distinct those of the CXC chemokines, and the present implication of the third extracellular domain might reflect this difference; neutralizing antibodies to other CC receptors must be tested before formal conclusions can be reached. Second is the surprising fact that amino terminal-specific mAb, for example MCP R-02, can mimic the chemokine response. This allows us to consider this region critical in agonist activation of the CCR2 receptor.
We therefore conclude that chemokine receptors are organized into two distinct functional domains, corresponding to the NH.sub.2 terminal and third extracellular loop regions. All known chemokine receptors have a high degree of sequence identity and many chemokines can interact with more than one chemokine receptor. It is thus probable that ligand-receptor interactions involve similar regions in the distinct receptors, but effecting different modifications in the NH.sub.2 terminal domain implicated in signal transduction. Our findings could thus be extrapolated to the entire chemokine receptor family and contribute to an explanation of the degeneracy in chemokine-chemokine receptor signalling.
In the experimental part below we describe the generation of mAbs that are capable of recognizing native MCP-1 receptor, and their characterization including their ability to block and/or mimetize the MCP-1 and induction of Ca.sup.2+ in human monocytes and monocytic cell lines and chemotaxis.